title: DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation.
abstract: Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100-1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Minakshi
authors.initials: M
authors.lastname: Guha
authors.forename: Elena
authors.initials: E
authors.lastname: Castellanos-Rizaldos
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000483:Alleles; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy; D008297:Male; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011799:Quinazolines / Q000627:therapeutic use; D017422:Sequence Analysis, DNA
chemical_list: D004273:DNA, Neoplasm; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D011799:Quinazolines; C477371:locked nucleic acid; D066246:ErbB Receptors; D000077156:Gefitinib